Molecular markers of infection by parasitic organisms of major public-health importance, are being developed by us in order to enable a new approach for large-scale monitoring of their transmission.

Lymphatic filariasis, is currently subject to a worldwide eradication campaign initiated by the WHO. Post-control surveillance is carried out by monitoring the mosquito vectors for infection by the filarial parasites which cause the disease. Of these, Wuchereria bancrofti, the major disease-causing agent, infects about 110 million people in tropical areas. We have identified in the genome of this parasite a large dispersed repeated sequence we named LDR1, 1674 bp long , which exhibits characteristics of a region which attaches to the nuclear scaffold/matrix proteins (SAR/MAR). Certain sections of the LDR1 were selected by teams involved in eradication as targets for amplification by the PCR assay now used as a tool for post-control surveillance by detecting infected mosquitoes (reviewed in Weil GJ, Ramzy MR. 2007. Diagnostic tools for filariasis elimination programs. Trends in Parasitol. 23, 78-82).

Schistosomiasis control currently centers on large-scale drug treatment for reducing the typicalmorbidity features. Large-scale schistosomiasis control projects are in progress in Africa South-East Asia and South America. While the question of when should morbidity control be taken one step further towards transmission control and elimination is being debated, the long lasting control operations by drug treatment, often reinforced by snail control, have reduced transmission in certain areas to low endemicity, or even eliminated it completely, and this situation requires special monitoring efforts.

For identifying early changes in schistosomiasis transmission following control and before symptoms (self-reportable by questionnaires) re-appear, we have investigated the possibility of detecting man-to snail transmission by determining the prevalence of prepatent infections in the snail host. Since prepatent infection lasts for several weeks and represents snails penetrated by miracidia (including those where infection is not completed to cercarial shedding) it could be expected that the prevalence of prepatent infection, if detected from early prepatency onward, should be substantial as compared with cercarial shedding. This was indeed confirmed by us using the sensitive PCR for a large-scale monitoring of Schistosoma haematobium prepatent infection in snails collected from transmission sites in Coastal Kenya. Thus, while patent snail infection amounted to only about 1%, prepatent infection exceeded 30% indicating that much less snails are required for determining transmission potential by this approach, and that changes in prevalence of prepatent snail infection can be expected to detect early changes in man-to snail transmission resulting from reinfection. We have further demonstrated a statistically significant correspondence between prevalence of prepatent infection in snails and the prevalence and mean intensity of infection in the populations contacting the corresponding water-bodies/transmission sites, thus further suggesting the potential usefulness of screening of prepatent snail infection for assessing transmission.

For detecting prepatent schistosome infection in snails by PCR we undertook the identification of repeated DNA sequences in the genome of these parasites. The Sm1-7 sequence of S. mansoni is a 121 bp long repeat unit tandemly arranged in the schistosomal genome in large arrays constituting about 12% of the genome of this parasite, and thus allowing a very sensitive detection of infected snails from the first day after penetration of a miracidium. The detection sensitivity in this case is also very high. Likewise, a snail penetrated by a miracidium of S. haematobium can be easily detected by PCR amplifying the DraI repeated sequence, a 121 bp long sequence constituting about 15% of its genome. In the latter case, however, related schistosomes (mostly of animals) which also infect bulinid snail hosts of S. haematobium can be also detected by amplification of the DraI repeat. We therefore sought to differentiate S. haematobium from related animal schistosmes. This was recently accomplished by us using amplification of an inter-repeat sequence located between a newly identified S . haematobium repeated sequence, we named the Sh110 sequence, and the known schistosomal splice-leader sequence. Thus, PCR employing one primer of the sh110 sequence and a second one of the slice-leader sequence can differentiate S. haematobium from S. bovis, S. mattheei, S. intercalatum and S. currassoni (but not S. margrebowiei), and this should enable specific detection of S. haematobium-infected snails in most endemic areas.

Hydatidosis or cystic echinococcosis, caused in man and farm animals by cystic larvae of the dog tapeworm Echinococcus granulosus, is of cosmopolitan distribution with very high endemicity in the Mediterranean basin. Monitoring of transmission by examining dogs' feces for presence of eggs is impractical given that numerous other tapeworms shed eggs with identical morphology. Identification of worms following chemical purgation is also impractical for large-scale monitoring of transmission. Coprodiagnosis of the parasite's antigens lacks sufficient sensitivity and specificity. We have identified in the genome of E. granulosus a repeated sequence the HhaIII repeat tandemly arranged in units of 264 bp. It constitutes a few percent of the genome but its amplification using PCR enables very sensitive detection of infected dogs given that the assay can detect a single egg.

Toxoplasmosis is a protozoan infection widely distributed worldwide in many vertebrates including man and farm animals. It causes severe morbidity in fetuses of previously unexposed (and therefore immunologically naïve) mothers. In cats, the definitive host, the parasite Toxoplasma gondii, develops sexually in the intestines, and the resulting infective oocysts are excreted in the feces. Screening of infected cats is traditionally done by isolating and counting oocysts from feces by Microscopical examination, or by bioassay involving infection of mice. We have developed a copro-PCR based on amplification of a previously known repeated sequence 529 bp long. The assay enables both sensitive detection and differentiation of the parasite from other parasites appearing in feces with similar morphology. This assay should now enable replacing oocyte detection, an insensitive assay, and bioassay, a cumbersome, expensive and time consuming assay, for large-scale monitoring of transmission by cats.

Although PCR has already been used for large scale screening of parasitic infection (lymphatic filariasis and schistosomiasis), the full potential of molecular monitoring for large-scale evaluation of transmission should greatly benefit from adapting newer DNA amplification methods that are technically simple and therefore applicable in field laboratories. Also, DNA extraction from fecal material can be simplified for large-scale monitoring by copro- PCR. Our forthcoming work centers on adaptation of existing molecular monitoring methods we have already developed, to actual fieldsituations.